To assess the functional importance of overall ITAM numbers, as well as the contribution of individual CD3 ITAMs in vivo, the authors generated 25 groups of mice, each expressing a TCR complex with a different combination of wild-type and mutant CD3 ITAMs. To achieve this, 'retrogenic mice' were generated by reconstituting Rag1−/− mice (which are deficient in the recombination-activating gene 1 and therefore lack lymphocytes) with bone marrow transduced with a retrovirus encoding either wild-type CD3 chains or CD3 chains containing various combinations of ITAM mutations. As the donor bone marrow was from mice that lacked expression of endogenous CD3 proteins, the functionality of retrovirally expressed CD3 chains that contained the entire range of 1–10 ITAMs could be assessed in the recipient mice.
Mice that received bone marrow transduced with Cd3 containing 2–6 functional CD3 ITAMs developed a lethal multi-organ autoimmune syndrome, whereas mice that expressed 7 or more CD3 ITAMs remained healthy. The autoimmunity in the diseased mice was mediated mainly by CD4+ T cells and in some mice was accompanied by the production of autoantibodies. Closer examination of the diseased mice revealed that their populations of forkhead box P3 (FOXP3)-expressing regulatory T cells were intact, whereas negative selection was impaired, meaning that large numbers of autoreactive T cells escaped from the thymus into the periphery. So, a breakdown in central rather than peripheral tolerance mechanisms led to autoimmunity in mice with less than 7 functional CD3 ITAMs, and relatively subtle changes in TCR-signalling strength resulted in defective negative selection of autoreactive T cells.
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